These practical protocols are designed for students doing an Extended Project Qualification, Advanced Higher investigation, or IB investigation in biology.
They are linked to our Cutting the world’s food waste project starter.
Areas for investigation
Given the enormous biochemical and structural changes that occur during ripening, it would be very surprising if the water potential and biomechanical properties of the fruit or vegetable did not change.
Some possible investigations include:
- the changes in starch and / or glucose concentration during ripening
- how the water potential changes during ripening
- how the water potential changes during storage or drying out
- how the stiffness of the fruit changes during ripening
- how the stiffness of the fruit changes during storage or drying out
- the relationship, if any, between polygalacturonase activity and firmness
A method for measuring the water potential of some fruit or vegetable tissues
This procedure involves placing weighed samples of plant tissue in solutions of varying water potential, and then re-weighing them after an number of hours. This can be done successfully only with fairly solid tissues, such as apple and potato, which can be cut with a cork borer. By plotting a graph of the gain or loss of mass against the concentration of the bathing solution you can determine the water potential of the plant tissue cells.
This technique depends upon the effects of osmosis between the tissue sample and the solution that bathes it. If the water potential of the tissue is greater than that of the bathing solution, then water is lost from the tissue, and this is reflected as a loss in tissue mass. If, however, the water potential of the tissue is less than that of the bathing solution, then the tissue gains water and this is reflected as an increase in the mass of the tissue.
- Use a cork borer to remove a series of cylinders of tissue from the fruit or vegetable. The cylinders should be cut of the same diameter (e.g. 1 cm), and from comparable regions of the plant organ.
- Place the cylinders on a tile and use a scalpel or razor to remove any remaining skin. Then carefully cut them to exactly the same length (e.g. 2 cm).
- Make up a series of bathing solutions of different concentrations, say 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 0.7 and 1.0 M sucrose in water.
- Weigh each cylinder and place in a labelled test tube or boiling tube with sufficient bathing solution to cover the cylinder to a depth of at least 5 cm.
- Leave the cylinders to soak for several hours, or better still, overnight in a refrigerator (to avoid the growth of microbes in the sucrose solution).
- Remove each cylinder from its bathing solution. Blot briefly on a paper towel then re-weigh.
- Calculate the loss or gain in mass by the cylinders. Plot a graph with the loss / gain of mass on the y-axis and sucrose concentration on the x-axis.
From the trend of data points it should be possible to interpolate the concentration at which no gain or loss of mass might have been expected to occur – at this (theoretical) concentration, the water potential of the bathing solution is equivalent to that of the tissue.
As an alternative, you could shorten the period for soaking by cutting the cylinders into a series of slices approximately 1 mm thick, and then placing them on a long steel pin, so that they are all separated by a small space. These pins are then immersed in the sucrose solutions as described above. The osmotic equilibrium occurs within an hour or so.
Some vegetables may have water potentials outside those detectable by the range suggested above, so pilot experiments may be needed. Also, for more detailed measurements, additional concentrations of sucrose may be needed within the range suggested.
Biomechanical tests on ripening fruits and vegetables
Squeeze most fruit, and you’ll feel how soft and ripe it is. But how might softness be quantified? In addition, it is always easier to peel a ripe fruit. How can we measure the ease of peeling?
Testing a fruit’s softness using a compression test
- This depends on the principle that, when a load is applied, softer fruits undergo greater distortions in shape than harder ones.
- Place the fruit on a level, hard surface and arrange for a simple light platform to rest on its upper surface. A thin piece of stiff aluminium sheet would be a suitable material for the platform. Measure the distance between the platform and the surface on which the fruit is placed.
- Apply a known mass to the platform then re-measure the distance between the platform and surface. Calculate the change as a result of adding the mass.
- It is important that the mass is not too heavy, so that it causes rupture of the fruit. Some trial and error, therefore, is needed to judge what mass works best. If the mass is distorting the fruit within its elastic limit, then after removal of the mass the fruit should recover its shape.
- It would be best to take measurements within these elastic limits, as the tissue ‘failure’ involved in more extreme distortions make the biomechanical comparisons too complex to resolve.
- It may be sufficient to carry out this test with a single mass, and then make comparisons between fruits at different stages of ripeness. Precautions need to be taken to control factors such as orientation of the fruit (take several measurements, with the fruit in different orientations) and the size of the fruit (express the distortion as a percentage).
- A further elaboration of this technique would be to add successive loads, then measure the increase in distortion, until the eventual ‘failure’ of the tissues. Plot the percentage distortion versus applied load – the gradient reflects the stiffness of the tissue (but only within the elastic limits of the fruit).
Testing a fruit’s softness using a penetration test
This is based on the principle that it is easier to drive a nail into wood than into concrete!
- Peel the skin from the fruit to be tested. Then prepare a block of the fruit to be tested and place it under the ‘penetration device’. This might be anything, ranging from a mounted needle to a sharpened pencil. Support the device by some kind of sleeve which allows vertical movement.
- Apply a load to the device, causing it to penetrate the flesh of the fruit. Find the size of an appropriate load by trial and error. The load could be applied by a force meter, thus allowing detailed information about the force applied! Alternatively, use known masses applied to the penetration device.
- Measure the extent of penetration of the flesh by the device. (This could be made easier by marking its sides with mm scale).
Possible complications include the fact that the flesh may vary in strength according to the type of tissue being penetrated. Also, if serious fractures develop in the fruit, then penetration may be the result of the propagation of cracks rather than loosening of cell walls.
By measuring the force necessary to detach a piece of skin, the ease of peeling can be assayed.
- Use a scalpel to cut completely through the skin of the fruit, forming a 2 cm square. Leave the square in contact with the fruit flesh beneath.
- Use ‘Superglue’ to fix a metal tag to the square (or perhaps attach a fish-hook to the epidermal layers).
- Attach a force meter to the metal tag and gradually increase the force until the square of skin pulls loose. Note the force involved.
- Repeat these measurements with some other areas of the fruit, to obtain a mean value.
Some ‘biotechnological tricks’ have been played with fruits, such as injecting pectinases under the skin of an orange to cause the skin to be more easily removed. The techniques described above might offer a way of quantifying accurately any changes in the ease of peeling.
Thickness of skin
In fruits such as the banana, the thickness of the skin undergoes a noticeable change during the ripening process. Perhaps this could be quantified, and related to the changes in the strength of skin attachment to the underlying flesh. There may be other macro or microscopic changes in structure that could be followed during the ripening process.
Pectins consist of a very diverse range of water-soluble polysaccharides, such as homogalacturan, rhamnogalacturan and arabinogalacturan. Pectins are the most ‘hydrophilic’ components of the cell wall and they are insoluble in alcoholic solutions.
The principle of this assay is to boil the fruit (or vegetable) in alcohol to remove most of the alcohol-soluble carbohydrates of the cells, and then to boil in water followed by alcohol-precipitation of the pectins.
- Cut the fruit (or vegetable) into thin slices (no more than a few mm thick), and place a weighed amount into boiling industrial methylated spirits (IMS) in a boiling tube. The volume of IMS should be approximately 5 cm3 per g of fruit.
Safety note – Inflammable solution – heating must be done using a thermostatic water bath, NOT a naked flame.
- Boil in IMS for at least 5 minutes, then dispose of the IMS safely.
- Place the slices in a mortar and grind with silver sand and distilled water to form a smooth, loose slurry. Depending upon amounts being prepared, a blender could be used.
- Decant the slurry into a glass beaker and make the volume to a measured total (e.g. 150 cm3 for an initial mass of fruit = 50 g). Boil the mixture for 10 minutes, using a hot-plate (or bunsen flame only if no boiling ethanol is in laboratory). Pay careful attention to the boiling mixture, to avoid it foaming over the sides of the beaker.
- After boiling, cool the beaker to room temperature and add a little more water to restore any volume evaporated during boiling.
- Use a pH meter in the slurry and add 1M NH4OH dropwise until the mixture is pH 6.5.
- Separate the solids using a Buchner funnel or centrifuge. Dispose of the solids but keep the liquid extract (which contains the dissolved pectins).
- Mix the pectin solution with ethanol in the ratio 1 part pectin : 4 parts ethanol. The pectins should now precipitate out of solution.
- Filter off the pectins, or better still, centrifuge the mixture. Then dispose of the supernatant and keep the pectin pellet, which is either weighed wet, or dried to a constant mass.
- The pectins thus extracted can have their identity checked by being re-dissolved in water. Pectins form a milky solution, and, when pectinase is added, the milkiness clears after incubation, say at 35 °C for one hour.